Application Of Nucleic Acid Aptamer-Functionalized Magnetic Beads In The Separation And Detection Of Fumonisin B1

Fumonisin(FB)is a carcinogenic mycotoxin produced by the genus Fusarium,which mainly contaminates corn and other grains and their products.FB1 is the most common and the highest proportion of FB,and is classified as a class 2B carcinogen by the International Agency for Research on Cancer(IARC)due to its strong toxicity.The current methods for detecting FB1 are mainly instrumental methods and immunological methods,but there are disadvantages such as complicated operation,high cost,interference by food matrix or low sensitivity.Therefore,it is of great significance to establish an accurate,low-cost and strong anti-matrix interference detection method for FB1.In recent years,magnetic separation,as a sample pretreatment technology,has been gradually applied to the pretreatment of toxin samples.This technology has the advantages of simple operation,elimination of food matrix interference,and improved sensitivity.In this study,aptamer(Apt)was used as the recognition molecule,and polyethylene glycol(PEG)was introduced as a mediator to reduce steric hindrance and increase aptamer loading to achieve higher recognition.Efficiency,preparation of PEG-mediated aptamer-functionalized magnetic beads(Apt-PEG-MBs),combined with Ultra Performance Liquid Chromatography(UPLC),colorimetry and fluorescence methods,to establish methods for enrichment and detection of FB1 in corn flour.The contents of each chapter are described as follows:The first chapter introduces the detection method of FB1,and reviews the research progress of nucleic acid aptamer-functionalized magnetic beads in the detection of mycotoxins.In the second chapter,to realize the enrichment,separation and detection of FB1 in corn flour,a method based on Apt-PEG-MBs combined with UPLC to detect FB1 was established.In this study,using aptamers as recognition molecules and PEG as mediators,Apt-PEG-MBs were synthesized,which can specifically bind to the target toxin FB1 to obtain FB1@Apt-PEG-MBs complexes.The compound was heated at 90~oC for 10 min,FB1 was dissociated from the FB1@Apt-PEG-MBs complex,and finally FB1 was detected by UPLC.Under the optimal conditions,the limit of detection(LOD)and limit of quantitation(LOQ)of this method for FB1 in phosphate-buffered saline(PBS)were 1.25 ng/m L,3.50 ng/m L,the LOD and LOQ of FB1 in the spiked corn flour samples were 3.75 ng/m L and 8.20 ng/m L,respectively.The research shows that the established method can be used for the detection of FB1 in corn flour,and the method has high accuracy and good specificity.In the third chapter,in order to realize the rapid and sensitive detection of FB1in corn flour,a method based on Apt-PEG-MBs combined with tyramine signal amplification(TSA)was established to detect FB1.First,biotin-modified DNA(Biotin-c DNA)that is partially complementary to the aptamer is put in,which can bind to the aptamer on Apt-PEG-MBs to form the c DNA@Apt-PEG-MBs,FB1 was added to compete for the c DNA binding sites,and after magnetic separation,streptavidin-horseradish peroxidase(SA-HRP)was added to and combined with the remaining c DNA on Apt-PEG-MBs by biotin-avidin system(BAS).Then TSA technology was introduced to realize signal amplification.Under the optimal experimental conditions,the LOD of Apt-PEG-MBs combined with common colorimetry to FB1 in PBS and spiked corn flour samples were 0.49 ng/m L and 0.61ng/m L,respectively,and the specificity was good.The LOD of Apt-PEG-MBs combined with TSA colorimetry to FB1 in PBS and spiked corn flour samples were0.02 ng/m L and 0.03 ng/m L,respectively.The research shows that the established method can be used for the detection of FB1 in spiked corn meal samples,and the sensitivity has been improved.In chapter four,to further improve the sensitivity of detecting FB1 in corn flour,a fluorescent sensor based on Apt-PEG-MBs and roll circle amplification(RCA)technology was established and used for the detection of FB1.First,the DNA that is partially complementary to the aptamer is added to form a c DNA@Apt-PEG-MBs complex,and then FB1 is added to compete for the c DNA binding site,and the free c DNA under the competition is put into the RCA reaction system,which can be used as a primer to initiate RCA reaction and produces a large number of RCA products capable of forming G-quadruplex structures.Then,thioflavin T(Th T)was added to the system,Th T intercalated into the G-quadruplex,and the fluorescence signal was enhanced,thereby realizing the fluorescence detection of FB1.The results showed that the LOD of this method for FB1 in PBS and spiked corn flour samples were0.011 ng/m L and 0.014 ng/m L,respectively,and the method had good specificity.The research shows that the established fluorescence sensor can be used for the rapid detection of FB1 in spiked corn flour samples,and the fluorescence sensor has the advantages of high sensitivity and convenience.To sum up,this study established three methods for the detection of FB1 based on Apt-PEG-MBs combined with UPLC method,colorimetric method and fluorescence method,and evaluated them with corn flour spiked samples.The results show that the methods established for FB1 have the advantages of accuracy,good specificity or high sensitivity,and can be used for the detection of FB1 in corn flour spiked samples,providing a reference for the rapid separation and detection of mycotoxins in my country.